Modified siphons for improving metering precision

ABSTRACT

The present invention provides centrifugal rotors for delivering a premeasured volume of liquid to a chamber in the rotor. In particular the rotors comprise siphons for delivering a premeasured volume of liquid between a first and a second chamber in the rotor. The siphons of the invention are designed such that the inlet of the siphon on the first chamber is radially outward of the siphon outlet on the second chamber. The first chamber is emptied to a level equivalent to the radial position of the siphon outlet.

[0001] The present application is a continuation of U.S. patentapplication Ser. No. 08/562,327, which is a continuation of U.S. patentapplication Ser. No. 08/254,406, which is a continuation in part of U.S.patent application Ser. No. 08/115,162, each of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to devices and methodsfor analyzing biological fluids. In particular, it relates to the designand use of improved centrifugal rotors having siphons which allowdelivery of a precise volume of liquid to a chamber in the rotor.

[0003] Biological tests of blood plasma and other biological fluidsfrequently require that fluids be quickly divided into predeterminedvolumes for analysis in a variety of optical tests or assays. It is alsofrequently desirable to separate potentially interfering cellularcomponents of the material from the other fluid prior to testing. Suchmeasurement and separation steps have previously been typicallyperformed by centrifugation to separate, for instance, blood plasma fromthe cellular components, followed by manual or automated pipetting ofpredetermined volumes of the blood plasma into separate test walls. Suchprocedures are labor intensive and time-consuming. As a result, variousautomated systems and methods have been proposed for providing multiplealiquots of plasma suitable for testing in a more efficient manner.

[0004] A major advance in the analysis of biological fluids has been theuse of centrifugal rotors. These rotors are designed to measure volumesof a biological fluid, such as blood, remove cellular components, andmix the fluid with an appropriate diluents for analysis, for example byoptical testing. Typically, the rotors provide a plurality of discretevolumes of sample in separate cuvettes in which the sample is opticallyanalyzed.

[0005] To ensure accurate and consistent results, such rotors requirethe delivery of precisely measured volumes of liquid to various chambersin the rotor. This must often be accomplished in circumstances in whichthe rotor quickly accelerates and decelerates or is otherwise perturbedduring operation. This perturbation can often lead to delivery ofinaccurately measured volumes. The present invention addresses these andother needs.

DESCRIPTION OF THE BACKGROUND ART

[0006] U.S. Pat. Nos. 4,894,204 and 5,160,702 disclose siphons fortransferring fluids between chambers in a rotor. U.S. Pat. No. 4,244,916discloses a rotor comprising a plurality of cuvettes positioned radiallyoutward of a central receptacle. Each cuvette is connected to thecentral receptacle by a duct and comprises a separate air escapeorifice. U.S. Pat. No. 4,314,968 relates to rotors having cellspositioned on the periphery of the rotor. Each cell includes aperipheral orifice for removing fluid introduced into the cell. U.S.Pat. No. 4,902,479 discloses a multicuvette rotor comprising elongated,radially extending cuvettes. Each elongated cuvette comprises a firstchamber for receiving a first constituent and a second chamber forreceiving a second constituent. A divider structure between the firstand second chambers prevents mixing of the constituents before apredetermined time. Mixing occurs as the rotor is spun at a significantspeed. U.S. Pat. No. 4,963,498 discloses devices which rely uponcapillaries, chambers, and orifices to pump and mix fluids for opticalanalysis. U.S. Pat. No. 5,077,013 discloses rotors comprising peripheralcuvettes connected to holding chambers positioned radially inward fromthe cuvettes.

SUMMARY OF THE INVENTION

[0007] The present invention provides centrifugal rotors comprisingsiphons for delivering a premeasured volume of liquid, typically abiological sample such as plasma, between a first and a second chamberin the rotor. The siphons of the invention have an elbow that isradially inward of the radially most inward point of the fluid in thefirst chamber. As the rotor is spinning the fluid does not flow past theelbow. After the rotor stops, capillary forces “prime” the siphon bypulling fluid just around the elbow. When the rotor is restarted,centrifugal force draws the remaining fluid out of the metering chamberinto the receiving chamber until the level of the fluid in the meteringchamber is at the same radial distance as the outlet of the siphon. Thesiphons of the invention are designed such that the inlet of the siphonon the first chamber is radially outward of the siphon outlet on thesecond chamber.

[0008] The positioning of the inlets and outlets of the siphons of theinvention provide a number of advantages. For example, the inlet of thesiphon is always positioned radially outward of the final position ofthe meniscus of the fluid in the first chamber, after fluid has beentransferred to the second chamber. Thus, inaccuracy in measurementassociated with different shaped menisci in different fluids isminimized since the meniscus is minimized. In addition, one of skillwill recognize that all siphons are semi-stable because the train offluid in a siphon is stable but easily broken if the rotor is perturbed.When the train of fluid is broken, under centrifugal force, the fluidcontained in the siphon will flow to the radially most outward point. Inprior art siphons this point is the siphon outlet. Thus, the potentialexists for the delivery of unmetered volumes of fluid to the receivingchamber. In the siphons of the present invention, the radially mostoutward point in the siphon is the siphon inlet. In this design, theproblem of delivering unmetered volumes of fluid is avoided because thefluid flows back into the first chamber when the train of fluid isbroken.

[0009] The chambers connected by the siphons of the invention are usedto perform any of a number of functions, such as metering liquids,separating solid components from a sample, mixing diluent with thesample, and the like. In the preferred embodiments, the siphons connecta plasma metering chamber to a mixing chamber for mixing the premeasuredvolume of plasma with diluent.

[0010] In addition, the rotors of the invention comprise unmodifiedinlet channels connecting a distribution ring to cuvettes comprisingreagents for optical analysis of a biological sample. The inlet channelsare sized such that, as the rotor spins, gas escapes from the cuvettethrough the inlet channel as the liquid enters the cuvette through theinlet channel. An “unmodified inlet channel” as used herein refers to asimple inlet channel, typically having a rectangular cross section,which is not modified (e.g., by altering the cross-sectional shape,surface texture, and the like) to provide a pathway for gas to escapefrom a cuvette that is not otherwise vented.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1A-1F are top plan views of a rotor of the inventionshowing the flow of fluids through the chambers and channels of therotor as the rotor is spun.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0012] The present invention provides methods and devices for thedelivery of liquids to chambers in an analytical rotor. The rotors ofthe invention comprise siphons which ensure precise delivery of meteredvolumes of liquid to a desired chamber in the rotor.

[0013] The rotors of the invention are suitable for the analysis of anyliquid, typically a biological sample such as whole blood or plasma. Itis also useful with numerous other biological fluids, such as urine,sputum, semen, saliva, ocular lens fluid, cerebral fluid, spinal fluid,amniotic fluid. Other fluids that can be tested include tissue culturemedia, food and industrial chemicals, environmental samples and thelike.

[0014] The rotors typically provide chambers which can separate cellularcomponents from the biological sample (e.g. whole blood), measure aprecise volume of liquid sample (e.g. plasma), mix the sample with anappropriate diluent and deliver the diluted sample to cuvettes foroptical analysis. The fluid delivered to the cuvettes, undergoesreaction(s) within the cuvettes, e.g., reaction with a reagent whichforms part of an analytical procedure to detect one or more analyteswithin the fluid. The sample may further be optically analyzed whilepresent in the rotor, either with or without prior reaction.

[0015] The apparatus of the present invention comprises an analyticalrotor having a rotor body which is capable of being mounted on aconventional laboratory centrifuge of the type which is commerciallyavailable from suppliers, such as Beckman Instruments, Inc., SpincoDivision, Fullerton, Calif.; Fisher scientific, Pittsburgh, Pa.; VWRScientific, San Francisco, Calif., and the like. Generally, thecentrifugal rotor will include a receptacle or other coupling devicesuitable for mounting on a vertical drive shaft provided by thecentrifuge. The particular design of the receptacle or coupling devicewill depend on the nature of the centrifuge, and it will be appreciatedthat the centrifugal rotor of the present invention may be adapted foruse with all or most types of centrifuges which are now available orwhich may become available in the future.

[0016] The rotor body comprises a structure which maintains a desiredgeometric pattern or relationship between a plurality of chambers,interconnection passages, and vents, as described in more detail below.Various specialized chambers and channels suitable for use in the rotorsof the invention are disclosed in U.S. Pat. Nos. 5,061,381 and5,122,284, and U.S. Ser. Nos. 07/678,762 and 07/783,041 which areincorporated herein by reference.

[0017] Usually, the body will be a substantially solid plate or diskwith the chambers and passages formed as spaces or voids in theotherwise solid matrix. Conveniently, such solid plate structures may beformed by laminating a plurality of separately-formed layers togetherinto a composite structure where the chambers and horizontal passagesare generally formed between adjacent layers. The vertical passages maybe formed through the layers. The individual layers may be formed byinjection molding, machining, or combinations thereof, and will usuallybe joined together, typically using a suitable adhesive or by ultrasonicwelding. The final enclosed volumes are formed when the layers arebrought together.

[0018] Of course, the centrifugal rotor could also be formed as aplurality of discrete components, such as tubes, vessels, chambers,etc., arranged in a suitable framework. Such assemblies of discretecomponents, however, are generally more difficult to manufacture and aretherefore less desirable than those formed within a substantially solidplate.

[0019] The rotor body may be formed from a wide variety of materials andmay optionally include two or more materials. Usually, the material(s)will be transparent so that the presence and distribution of thebiological fluid, cellular components, and reagents may be observedwithin the various internal chambers and passages. Optionally, to theextent analytical chambers, e.g., cuvettes, or other test wells areformed within the rotor, it is desirable to have suitable optical pathsformed within the rotor so that the contents of the cuvettes may beobserved spectrophotometrically, fluorometrically, or by other opticalassessment instruments. The construction of suitable cuvettes havingparticular optical paths formed therethrough is disclosed in U.S. Pat.No. 5,173,193, the disclosure of which is incorporated herein byreference. In the preferred embodiment, the rotor is formed with anacrylic resin having suitable optical properties, at least in thoseareas which define an optical path.

[0020] The apparatus and method of the present invention are suitablefor performing a wide variety of analytic procedures and assays whichare beneficially or necessarily performed on blood plasma and othersamples. The analytic procedures may require that the sample be combinedwith one or more reagents so that some detectable change occurs whichmay be related to the presence and/or amount of a particular component(analyte) or characteristic of the sample. For instance, the sample mayundergo a reaction or other change which results in a change in color,fluorescence, luminescence, or the like, which may be measured byconventional spectrophotometers, fluorometers, light detectors, and thelike. In some cases, immunoassays and other specific binding assays maybe performed within the cell-free fluid collection chamber or withincuvettes which are connected to the collection chamber. Generally, suchassay procedures should be homogeneous and not require a separationstep. In other cases, however, it may be possible to accommodateheterogeneous assay systems by providing a means to separate the sample(e.g., blood plasma) from the collection chamber or another test well orcuvette after the immunological reaction step has occurred. One of skillwill recognize that the means of analyzing the sample is not animportant aspect of the invention. Any of a number of analytical methodscan be adapted for use in the rotors of the invention, depending uponthe particular sample being analyzed and component being detected.

[0021] In the case of blood analyses, conventional blood assays aretypically performed. Examples of assays which may be performed includethose designed to detect glucose, lactate, dehydrogenase, serumglutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvictransaminase (SGPT), blood urea nitrogen (BUN), total protein,alkalinity, phosphatase, bilirubin, calcium, chloride, sodium,potassium, magnesium, and the like. This list is not exhaustive and isintended merely as being exemplary of the assays which may be performedusing the apparatus and method of the present invention. Usually, thesetests will require that the blood and plasma be combined with one ormore reagents which result in an optically detectable, usuallyphotometrically detectable, change in the plasma. The reagents which arerequired are well known and amply described in the patent and scientificliterature.

[0022] The reagents are preferably provided in lyophilized form toincrease stability. Ideally, they are provided in the form oflyophilized reagent spheres as described in U.S. Ser. No. 07/747,179,which is incorporated herein by reference.

[0023] Referring now to FIGS. 1A-1F, an analytical rotor comprising thechambers and channels of the present invention can be seen. FIG. 1Ashows the position of a blood sample 102 in the blood applicationchamber 104 after the sample has been loaded in the rotor body 100. Adiluent container in chamber 106 is opened upon mounting of the rotor onthe spindle of the centrifuge as described in copending and commonlyassigned application, U.S. Ser. No. 07/873,327, which is incorporatedherein by reference.

[0024]FIG. 1B shows the position of the diluent 108 and blood sample 102after the rotor is spun at 4,000 rpm. The blood sample 102 begins toexit the blood application chamber 104 and enters the plasma meteringchamber 110. At the same time, diluent 108 empties from the diluentcontainer into the holding chamber 112. The diluent immediately beginsto enter the diluent metering chamber 114 through channel 116.

[0025]FIG. 1C shows the position of the liquids as the rotor 100continues to spin. Here, the blood sample 102 has emptied the bloodapplication chamber 104 and overflows the plasma metering chamber 110into the overflow chamber 118 where it flows to the hemoglobin cuvette120 and the excess blood dump 122. Meanwhile, diluent 108 fills thediluent metering chamber 114 and excess flows through channel 124 todiluent-only cuvettes 126 and excess diluent dump 127.

[0026]FIG. 1D shows the position of the liquids at the conclusion of thefirst spin. The blood sample 102 has separated into cells 128 and plasma130. The diluent-only cuvettes 126 are filled and a predetermined amountof diluent remains in the diluent metering chamber 114. The rotor 100 isthen stopped and the siphon 132 from the diluent metering chamber 114,as well as the siphon 134 from the plasma metering chamber 110, areallowed to prime, as described above. Siphon 134 is a siphon of thepresent invention. It is connected to the plasma metering chamber 110 atinlet 138. The inlet 138 is positioned radially outward of the siphonoutlet 139, through which the siphon 134 empties into the mixing chamber136.

[0027]FIG. 1E shows the position of the liquids during the second spinof the rotor. The diluent metering chamber 114 empties into the mixingchamber 136 through siphon 132. A predetermined amount of plasma 130 ismetered into the mixing chamber 136 and the two fluids are mixed,thereby forming diluted plasma 131. The amount of plasma 130 deliveredto the mixing chamber 136 is determined by the position of the outlet139 on the siphon 134. As can be seen in this figure, the final level ofthe plasma 133 in the plasma metering chamber 110 is at the same radialposition as the outlet 139. Thus, the volume of plasma delivered to themixing chamber 136 is determined by the volume of the plasma meteringchamber 110 between the exit to the overflow chamber 129 and the finallevel of plasma 133. After the plasma and diluent are mixed in themixing chamber 136, the rotor is stopped again and the output siphon 140is primed.

[0028]FIG. 1F shows the position of the diluted plasma 131 as the rotoris spun during the third spin. This figure illustrates the movement ofthe diluted plasma 131 through the distribution ring 142 and inletchannels 144 to the cuvettes 146 and excess plasma dump 147. Theresistance to flow in the output siphon 140 is selected to be higherthan the resistance to flow in the distribution ring 142 and the inletchannels 144 so that air present in the cuvettes 146 can escape as thecuvettes are filled. Specifically, siphon 140 is dimensioned such thatthe ratio of the cross sectional area of the inlet channels 144 to thecross sectional area of the liquid in them is greater than 2:1,preferably greater than about 4:1. The cross sectional area of the inletchannels 144 is typically the same as or slightly smaller than that ofthe distribution channel 142 so that gas in the unvented cuvettesescapes through the inlet channels 144 and distribution 142. If thesample is plasma or diluted plasma and the channels are rectangular incross-section, their dimensions are typically as follows: siphon: 0.150mm depth, 0.200 mm width; distribution channel 0.300 mm depth, 0.5 mmwidth; inlet channels: 0.150 depth, 0.500 width.

[0029] After the cuvettes have been filled, reagents present in thecuvettes are mixed with the solution and the necessary photometricanalyses are made on the sample. Such analyses are carried out asdescribed above according to methods known to those of skill in the art.

[0030] Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.

1. A centrifugal rotor comprising: a rotor body including: aliquid-dispensing chamber; a liquid-receiving chamber; and a siphonconnecting the liquid-dispensing chamber and the liquid-receivingchamber, said siphon including: a siphon inlet connected to theliquid-dispensing chamber; a siphon outlet connected to theliquid-receiving chamber; and a siphon body portion between said siphoninlet and said siphon outlet, the body portion extending radiallyinwardly and having an innermost portion that is radially inward of theinnermost portion of the liquid-dispensing chamber. 2 The rotor of claim1, wherein said liquid-dispensing chamber is a plasma metering chamberand said liquid-receiving chamber is a mixing chamber.
 3. The rotor ofclaim 1, wherein said liquid-dispensing chamber is a diluent meteringchamber and said liquid-receiving chamber is a mixing chamber.
 4. Therotor of claim 1, wherein said liquid-receiving chamber is adistribution ring.
 5. A centrifugal rotor comprising: a rotor bodycomprising a liquid-dispensing chamber containing a liquid, aliquid-receiving chamber, and a siphon; the siphon being connected tothe liquid-dispensing chamber through a siphon inlet and connected tothe liquid-receiving chamber through a siphon outlet, the siphon inletbeing radially inward of the siphon outlet, said siphon travelingradially inward to a point radially inward of said siphon inlet and thenradially outward to said siphon outlet; the rotor further comprising: acuvette containing reagents necessary for the analysis of a biologicalsample, wherein said cuvette is radially outward of saidliquid-dispensing chamber and said liquid-receiving chamber, and; adistribution ring which permits flow of a liquid to said cuvette from anoutput siphon connected to the liquid-receiving chamber.
 6. Acentrifugal rotor comprising: a rotor body comprising aliquid-dispensing chamber containing a liquid, a liquid-receivingchamber, and a siphon; the siphon being connected to theliquid-dispensing chamber through a siphon inlet and connected to theliquid-receiving chamber through a siphon outlet, the siphon inlet beingradially inward of the siphon outlet, said siphon traveling radiallyinward to a point radially inward of said siphon inlet and then radiallyoutward to said siphon outlet; the rotor further comprising: adistribution ring positioned radially outward of the liquid receivingchamber; and a delivery channel connecting the distribution ring to theliquid-receiving chamber, said distribution ring being connected to acuvette through an inlet channel.
 7. The rotor of claim 6, wherein theinlet channel has a cross sectional area at least about 1.5 times thecross sectional area of the delivery channel.
 8. The rotor of claim 7,wherein the cross sectional area of the inlet channel is about 2 timesthe cross sectional area of the delivery channel.
 9. The rotor of claim7, wherein the cross sectional area of the delivery channel is about0.03 mm².
 10. The rotor of claim 6, wherein the delivery channel is asiphon.
 11. A method of delivering a premeasured volume of liquid from afirst chamber to a second chamber in a rotor, the method comprising:providing a rotor comprising a first chamber with a first volume, asecond chamber, and a siphon connected to the first chamber through asiphon inlet and connected to the second chamber through a siphonoutlet, the siphon inlet being radially inward of the siphon outlet;spinning the rotor, thereby introducing an unmeasured volume of liquidinto the first chamber; stopping the rotation of the rotor, therebypriming the siphon connecting the first chamber to the second chamber;and spinning the rotor, thereby initiating the operation of the siphonand delivering the premeasured volume of the liquid from the firstchamber to the second chamber, the premeasured volume being determinedby the radial position of the siphon outlet and the first volume of thefirst chamber.